Generation of reactive oxygen species in a human keratinocyte cell line: role of calcium.
In the human keratinocyte cell line HaCaT, reactive oxygen species (ROS) were generated in a dose- and time-dependent manner in response to epidermal growth factor (EGF), bradykinin, thapsigargin, and the Ca(2+)-ionophore A23187, agonists that interact with different primary cell targets. ROS formation was assessed by both chemiluminescence- and fluorescence-based methods. The ROS evoked by EGF and bradykinin decayed within 8 and 4 min, respectively, this transient effect resulting probably from down-regulation of the specific agonist receptors or dissipation of the secondary signals. In contrast, the response to thapsigargin and A23187 was sustained for at least 15 min. Extracellular Ca2+ and a rise in intracellular Ca2+ concentration ([Ca2+]i) proved essential for ROS production. Chelation by BAPTA suppressed ROS formation. Direct measurement of [Ca2+]i using fura fluorescence revealed that EGF and bradykinin evoked a modest, transient [Ca2+]i elevation of less than twofold, whereas with thapsigargin and A23187 there was a sustained two- to fourfold elevation. For each agonist, the kinetics of the rise and decay of [Ca2+]i were similar to those of ROS. The enzyme(s) involved in ROS formation were inhibited by diphenyleneiodonium, indicating dependence on FAD. Our results suggest a close link between ROS and changes in [Ca2+]i generated by growth factors and hormones. This is a particularly interesting connection because elevation of ROS and/ or [Ca2+]i has been linked to cell proliferation, differentiation, and apoptosis.
Goldman
R
R
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.
Moshonov
S
S
Zor
U
U
D001713:Biphenyl Compounds / Q000494:pharmacology; D001920:Bradykinin / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D008163:Luminescent Measurements; D009861:Onium Compounds / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000819:agonists; D019284:Thapsigargin / Q000494:pharmacology
D001713:Biphenyl Compounds; D007476:Ionophores; D009861:Onium Compounds; D017382:Reactive Oxygen Species; D011956:Receptors, Cell Surface; C031291:diphenyliodonium; D005182:Flavin-Adenine Dinucleotide; D000001:Calcimycin; D004815:Epidermal Growth Factor; D019284:Thapsigargin; D006861:Hydrogen Peroxide; D001920:Bradykinin; D002118:Calcium